: This R21 grant application is based on the initial observation that bacterial plasmid DNA is spontaneously internalized in B lymphocytes. This event does not require the prior activation of cells, is very rapid, and is apparently conserved across species. I have also been able to preliminarily assess that a single injection of transgenic B lymphocytes in vivo elicits a T cell response against epitopes specificities coded by the gene contained in the plasmid. With these observations in hand there are many new issues that need to be addressed and many questions that beg for an answer. I have chosen to answer two in particular as they may relate to this newly discovered phenomenon in general terms and also pertain to current views on the interaction between bacterial DNA and cells of the immune system. Specifically, I will investigate the mechanism of spontaneous uptake of plasmid DNA by B lymphocytes testing the hypothesis that clathrin mediated endocytosis is responsible for B-cell uptake and utilization of plasmid DNA. The second question that I am interested in answering is whether or not once in the B lymphocyte plasmid DNA will activate the cell through TLR9, the intracellular endosome-associated receptor for CpG motifs in bacterial DNA. Since it is commonly accepted that CpG signal cells of the immune system constitutes a link between innate and adaptive immunity, my working hypothesis is designed to directly challenge this paradigm. I anticipate that in case the experiments prove that B cell activation and in vivo immunogenicity occur independently of CpG mediated TLR9 signaling, a logical conclusion will be that the current paradigm needs revision. Therefore, this application is based on two viable hypotheses and will have far reaching implications for future work with plasmid DNA in vaccination in humans.